Neuropeptide S (NPS) is a recently identified transmitter in the brain. NPS produces arousal and wakefulness and reduces behavioral signs of anxiety in rodents. Recent studies have also found that NPS stimulates drug-seeking behavior and enhances learning and memory. A naturally occurring mutation in the NPS receptor gene is associated with panic disorder, making the NPS system an interesting candidate for development of new drugs that could have therapeutic benefits in the treatment of anxiety disorders or psychiatric conditions involving improper processing of aversive memories, such as panic disorder, phobias or generalized anxiety disorder. However, it is currently unknown which other transmitters control the release of NPS in the brain and which transmitters are co-localized, and thus co-released, with NPS. To address these questions, we have generated transgenic mice that express a fluorescent marker protein (EGFP) in only those neurons that also synthesize NPS. These NPS/EGFP-transgenic mice have allowed us to collect single fluorescent neurons and determine their gene expression profile. Among all expressed genes, we are particularly interested in receptor genes as they indicate which incoming neurotransmitters may control activity of NPS-producing neurons. As a first result of these investigations, we have discovered functional receptors for the neuropeptide CRF, which is a key transmitter in mediating stress and anxiety behaviors. Furthermore, we have now a list of potentially co-localized other neurotransmitters that may be co-released whenever these neurons are activated. The first goal of the current proposal is to confirm the presence of these additional neurotransmitters in NPS-producing neurons by staining brain sections with suitable antibody probes. Secondly, we will study receptors located on NPS-producing cells that either activate or inhibit neuronal firing by recording their electrical activity in brain slices from NPS/EGFP-transgenic mice. As a proof of concept, we will focus on CRF receptors to establish the interaction of the NPS system with this major stress-regulating system. Finally, we will investigate if stress leads to activation of NPS neurons, and in particular neurons that express CRF receptors. This focused approach will allow us to understand functional responses and network interactions of the NPS system with the CRF system and could translate into novel therapeutic strategies for the treatment of anxiety or stress-related disorders.